| As the most widely used therapeutic protein drugs in clinical,antibodies are mainly expressed and produced by CHO(Chinese Hamster Ovary)cell culture.Compared with the previous generation of biotechnology products such as cell factor drugs,the clinical dosage of antibody is rather high.Therefore,antibodies need to be produced in industrial large scale to meet the market demands.And the process development,optimization and scale-up of CHO cell culture are rather challenging.Productivity improvement,cell culture scale enlargement and stable quality of antibodies become the main problems to be solved in the large-scale production of antibody protein drugs in the domestic antibody industry.Focusing on the above problems,this paper systematically studied the optimization of the fed-batch cell culture process of CHO cells in small scale,the scale up of the pilot and commercial scales and explored the enhanced concentrated fed-batch based on alternative tangential flow(ATF)technology,so as to further improve the scale productivity of antibody drugs in China.In this study,the effect of the concentration of metal ions in the medium on the quality of antibodies was investigated.When the concentration of copper ions in the medium was reduced from 1000 nM to 500 nM,the viable cell density of MAb A cell can be maintained at 1.2×107 vc/mL.Productivity remained unchanged,and the proportion of basic variants in charge variants decreased significantly.These results suggest that copper ions,as the cofactors of various enzymes,not only can maintain cell growth and metabolism,but also affect C-terminal proline amidation.When antibody MAb A cell was cultured in stirred reactor,dissolved oxygen was reduced from 60%to 20%.As a result,cell growth and antibody production were not significantly changed.The ratio of G0F in antibody N-glycosylation was significantly increased,while the ratio of G1F was decreased obviously,which indicating that dissolved oxygen level had a significant effect on galactose glycosylation.On the other hand,the increase of the proportion of acidic variants of antibody was observed when the culture time was prolonged.The main reason was that the free radicals and redox effects in the culture medium had certain effects on the physicochemical properties of antibody.Based on the gas-liquid mass transfer theory in mechanical stirred bubbling reactor,a set of animal cell reactor mass transfer model was developed.The model combines the bioreactor mass transfer properties and mammalian cell metabolic parameters to predict the cell culture process oxygen demands and carbon dioxide accumulation in bioreactors.This mass-balance model has been successfully applied during MAb A process one-step scaling up from 2-L to 1500-L bioreactor.The cell growth,antibody production and quality were consistent between the bench scale and commercial production scale.To solve the problem that it is difficult to further improve the antibody productivity significantly due to the accumulation of cell metabolic waste in the traditional CHO cell culture process,we developed a concentration fed-batch culture of CHO cells based on the alternative tangential flow(ATF)system.During the optimization of concentration perfusion culture process,the detection of free amino acid concentration in the medium played an important role,which provided a basis for the optimization of the perfusion medium component and the adjustment of perfusion rate.Finally,by concentrated fed-batch culture,the cell growth density of MAb A cells was increased by 7.5 times,and the antibody production was increased by 7.7 times. |
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